Reporter/marker genes have found wide use in the study of cellular genetic regulation and gene function. In studies of genetic regulation, a regulatory element (e.g. promoter or enhancer) fused to a reporter gene is transfected into cells. The amount of reporter molecule subsequently generated reflects the transcriptional activity of the regulatory element.
Reporter genes can be used to measure, among others, transcriptional activities of synthetic enhancers (e.g. enhancers formed by multimerization of a single nuclear binding site motif), and protein-protein interactions using two-hybrid systems.
In studies of gene function, marker molecules distinguish cells expressing transfected/infected genes from uninfected cells. A gene of interest is co-transfected with a marker gene. Cells expressing the gene of interest are identified by the presence of marker molecule. The marker molecule and the product of interest can be expressed as a protein fusion. Alternatively, the marker molecule and the product of interest can be expressed as distinct proteins from a transcriptional fusion having an internal ribosomal entry site (IRES).
With the recent development of reporter genes detectable by flow cytometry, it has become possible to analyze the expression of a transcriptional element within an individual mammalian cell. However, there is no currently available method of independently analyzing two transcriptional elements within a mammalian cell.
The articles by Nolan et al. in PNAS USA 85:2603-2607 (1985) and Fiering et al. in Cytometry 12: 291-301 (1991), herein incorporated by reference, describe FACS-Gal, a fluorogenic assay that permits the detection and isolation of individual cells expressing lacZ. The gene lacZ encodes the enzyme .beta.-galactosidase, which cleaves the non-fluorescent substrate fluorescein-di-.beta.-galactopyranoside to release fluorescein. For further information on FACS-Gal and some of its uses, see also the article by Kerr and Herzenberg in Methods: A Companion to Methods in Enzymology 2(3):261-271 (1991), herein incorporated by reference.
Another type of reporter gene previously used for FACS analysis of mammalian cells is described in an article by Rice et al. in PNAS USA 89(12):5467-5471 (1992). A gene encoding a plasma membrane surface receptor is transfected into cells that do not endogenously express the receptor. Fluorochrome-conjugated antibodies bind to receptor present on the cell surface, and can be detected by flow cytometry. Several difficulties have precluded the widespread use of receptor-encoding reporter genes, including the unquantitative nature of the detection, and the potential interference of the receptor with normal signal transduction pathways.
Recently, the green fluorescent protein (GFP) gene, isolated from the jellyfish Aequorea victoria, has become available as a potential reporter or marker in procaryotes and eucaryotes. The gfp gene encodes a protein which fluoresces when excited by violet or blue-green light. GFP is unique among reporters in that the GFP fluorophore spontaneously forms intracellularly without added cofactors, and in that it provides a direct readout of gene expression. The use of GFP eliminates the need for introducing a substrate into live cells, and for evaluating complex enzyme-substrate kinetics. For information on GFP see for example U.S. Pat. No. 5,491,084, herein incorporated by reference, and the articles by Peters et al. in Dev. Biol. 171:252-257 (1995), Rizzuto et al. in Curr. Biol. 5:635-642 (1995), and Cubitt et al. in Trends in Biochemical Sciences 20:448-455 (1995). The Cubitt et al. article includes a summary of reports of GFP expression in various procaryotic and eucaryotic systems.
GFP is a 238-amino acid protein, with amino-acids 65-67 (Ser, Tyr, and Gly, respectively) thought to be involved in the formation of the chromophore. For information on the structure of wtGFP, see the article by Yang et al. in Nature Biotechnology 14(10):1246-1251 (1996). FIG. 1-A shows a proposed biosynthetic scheme for the GFP chromophore. Only some of the expressed protein folds into a fluorescent form. For a given quantity of GFP within a cell, the GFP brightness depends on the quantum yield of the protein and on the fraction of protein correctly folded.
FIG. 1-B shows the excitation and emission spectra of wtGFP. Wild-type GFP has a major excitation peak at 395 nm, a minor excitation peak at 475 nm and a single emission peak at 509 nm. The two excitation peaks presumably reflect two distinct states (protonated and unprotonated) of the fluorophore. Illumination of wtGFP with UV or violet light results in photobleaching, and in photoisomerization from the state maximally excited at 395 nm to the state maximally excited at 475 nm. Photobleaching decreases the absolute magnitude of both excitation peaks, while photoisomerization reduces the relative magnitude of the major excitation peak. For data on photobleaching and photoisomerization of GFP, see the above-incorporated article by Cubitt et al.
A number of GFP mutants having altered excitation and emission spectra when expressed in E. coli are described in articles by Heim et al. in PNAS USA 91:12501-12504 (1994) and in Nature 373:663-664 (1995), herein incorporated by reference. Table 1 provides a summary of some characteristics of GFPs expressed in E. coli, as disclosed in the two Heim et al. articles.
TABLE 1 ______________________________________ Excitation Emission Mutation maximum maximum Relative fluorescence (%) ______________________________________ None 396 nm 508 nm =100 Ser-202 to Phe 398 nm 511 nm 117 (w/395 nm exc) Thr-203 to Ile Ile-167 to Val 471 nm 502 nm 166 (w/475 nm exc) Ile-167 to Thr 471 nm 502 nm 188 (w/475 nm exc) Tyr-66 to His 382 nm 448 nm 57 (w/395 nm exc) Tyr-66 to Trp 458 nm 480 nm Not done Ser-65 to Thr 489 nm 511 nm .about.600 Ser-65 to Cys 479 nm 507 nm .about.600 ______________________________________
Wild-type GFP as well as GFP mutants of various colors can be readily and quantitatively detected in bacteria, by fluorescence microscopy or flow cytometry. In mammalian cells, however, analysis of GFP fluorescence has proven relatively difficult, primarily because of the relatively high levels of autofluorescence in mammalian cells, and the high temperatures at which mammalian cells are grown. Molecules such as pyridine nucleotides, flavin nucleotides and flavoproteins fluoresce upon ultraviolet or blue excitation, and can obscure GFP fluorescence. Mammalian cell autofluorescence is particularly bright for UV excitation. Moreover, mammalian cells are typically grown at 37.degree. C., well above the temperature that yields maximal GFP fluorescence. The high temperatures at which mammalian cells are grown hinder the proper folding of GFP. For information on the temperature dependence of GFP fluorescence, see the article by Ogawa et al. in PNAS USA 92:11899-11903 (1995), herein incorporated by reference.
Wild-type or S65T GFP fluorescence can be qualitatively detected by flow cytometry in transiently transfected mammalian cells containing multiple copies of GFP expression vectors (data not shown, also reported in the article by Ropp et al. in Cytometry 21:309-317 (1995), herein incorporated by reference). However, the relative dullness of GFP in mammalian cells and the spectral overlap of wtGFP with available mutants have impeded the simultaneous detection by flow cytometry of multiple GFPs within an individual mammalian cell.
Ropp et al. describe an analysis by spectrofluorometry and flow cytometry of mammalian cells expressing wtGFP or S65T-GFP. The article also contains data on green autofluorescence for UV, violet, and blue green excitation in mammalian cells. The dullness of the described GFPs, and the similarity of the responses of wtGFP to blue and violet excitation would preclude the simultaneous flow cytometry analysis of wtGFP and S65T-GFP in mammalian cells. The two variants could not be used to distinguish cells expressing a single GFP variant from cells expressing both variants. Also, the two variants could not be used to independently analyze the expression of two transcriptional elements within mammalian cells.
Bender et al. (Bender, Kahana, Hudson, and Silver, personal communication) have isolated a number of mutants that show increased fluorescence in E. coli. In particular, one of the mutants (V163A) identified by Bender et al. retains the spectral properties (wavelengths of the excitation and emission peaks) of wild-type GFP but shows a 17-fold increase in fluorescence intensity in E. coli.